Olive Leaves (Olea Europaea L) Extract Loaded Lipid Nanoparticles: Optimization Of Processing Parameters By Box-Behnken Statistical Design, In-vitro Characterization, And Evaluation Of Anti-oxidant And Anti-microbial Activity Part 2

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2.5.6 Stability evaluation

The stability of optimized formulation(F9)was examined as per the International Council for harmonization(ICH)guidelines). SLN dispersion was taken in three separate glass vials(10 mL in each). Among the three vials, one was stored in the refrigerator(4℃±2℃), the second at room temperature(25±2℃/60±5 RH% RH), and the last one was stored in a stability chamber(Thermo Scientific, Sweden)at 40±2℃/75±5℃% RH. At a specific time point viz 1,3,6 month, the samples were taken out and examined for certain parameters like particle size, entrapment efficiency, zeta potential & PDI, etc, which were determined and compared statistically with zero time data(initial). 2.5.7 Antioxidant activity

One of the most useful methods for the detection of anti-oxidant potential is the 2,2-Diphenyl-1-Picryl-Hydrazyl-Hydrate free radicals(DPPH)method2. Radical scavenging activity(ie. analyzing their ability of scavenging DPPH free radicals)of OLP-SLNs was evaluated spectrophotometrically at 517 nm. Antioxidant activity of OLP, as well as OPL-SLNs, was determined with the help of(the DPPH)method. Solution of DPPH was prepared(0.1 mM)in ethanol. OLP-SLNs(equivalent to 0.2 mg/mL of OLP)were dispersed in phosphate buffer and left for 24 h to release the drug. An equivalent concentration of OLP(0.2 mg/mL)and blank SLNs was also prepared in the same medium. In this study, ascorbic acid of concentration 0.2 mg/mL was taken as a standard and while DPPH solution was used as a control. cistanche Australia 3300 μL DPPH was mixed in each solution of OLP-SLNs（500 μL）， blank SLNs， and OLP solution（500 μL）. Each reaction mixture was kept in a shaker water bath at 37±0.5℃ for 30 min under protected conditions from the light. Finally, the absorbance of each sample was determined by a UV-visible spectrophotometer(Shimadzu 1800, Japan)at 517 nm using ethanol was used as a blank. Each experiment was evaluated three times and values are presented as mean±SD. The value of anti-oxidant activity in the form of % DPPH scavenging activity was determined by the following formula(equation 2):

2.5.8 Anti-microbial study

The antimicrobial study of OLP-loaded SLNs(optimized batch F9)was conducted by utilizing the agar well diffusion method. Bacterial strains like Staphylococcus aureus (Gram's positive)and Pseudomonas aeruginoza(Gram's negative) were used for the evaluation ". Nutrient agar plates were prepared under sterile conditions and bacterial suspension（100 μL， concentration around 10°CFU/mL）of each bacterium was placed individually on each sterile nutrient agar plate with the help of sterile swabs incubated at 37±0.5℃ for 1 h. cistanche benefits Four wells around 8 mm diameters each were produced on each plate with the help of a sterile cork borer. Here, blank SLNs, positive control(pure extract), physical mixture(OLP extract+ blank), and SLNs and optimized OLP-SLNs preparation（100 μL）were used for the antimicrobial study and placed in each well. Plates were incubated at a temperature of 37±0.5℃. At predetermined time points(6,12,24 h), the plates were withdrawn and the zones of inhibition around the walls were measured in mm with the help of a caliper.

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3 Results and Discussion

3.1 Preliminary screening and pre-optimization of form-latent variables

The highest solubility of OLP was found to be in compri-to 888 ATO as depicted in Fig.1. The descending order of OLP solubility/100 gm in different lipid was compritol 188 ATO(36.93±4.92mg)>Precirol ATO 5(29.37±3.01 mg)>GMS(23.84±2.73mg)>palmitic acid(21.41±2.81 mg)>stearic acid(16.25±1.65 mg).Compritol 888 ATO had been used as a lipid for the preparation of oral-based SLNs for the preparation SLNs.

Based on the results of the preliminary screening study, tween 80 and compritol 888 ATO were selected as a surfactant and lipid, respectively. Other parameters like a drug to lipid ratio(1:3-1:6), surfactant concentration(%,1.5-4.5%)and homogenization speed (rpm, 3000-6000 rpm), homogenization time(2 h), sonication time(10 min)were decided by conducting the preliminary experiments. Factors like a drug to lipid ratio(A), surfactant concentration(B), and homogenization(C)were again optimized by using response surface methodology(RSM)in conjunction with 3-factors and 3-levels BBD, and their influence was detected on dependent factors viz particle size(Y1), entrapment efficiency(Y2)and PDI(Y3).

3.2 Preparation of SLNs and optimization of OLP loaded SLNS

The values of pre-optimized parameters as described

above were fitted to BBD of expert design. The composition of the total of seventeen formulations was explored by BBD with 5 center points. All formulations were developed and values of dependent parameters like particle size, entrapment efficiency, and PDI were examined and further fitted to BBD to get the final result. Polynomial equations and 3D graphs were generated showing the influence of independent factors on dependent factors. The positive sign of the polynomial equation explored a positive effect and the negative sign indicated a negative effect on dependent variables. Value of actual and predicted dependent variables regression analysis and Analysis of variance data of models are presented in Tables 1,2, and 3 respectively. The quadratic model was considered the best-fitted model for all responses as in this case the highest value of regression coefficient was observed.

3.3 Effect of certain parameters on particle size(Y1)

The following polynomial equation was obtained from the BBD indicating the effect of different independent factors on particle size(Y1):

The above-given equation exhibited that compritol 888 ATO showed a positive effect on the particle size while tween 80 and homogenization exhibited a negative influence on the particle size.

Here, variables A, B, C, AB, BC, and B" had a significant effect on the particle size. cistanche cholesterol At a 95% confidence interval, the lack of fit was insignificant(p>0.05)while the remaining parameters were found to be significant(p<0.0001)with adequate precision（>4）（Table 3）.Based on the R²

(0.9996)value, the quadratic model was considered as the best-fitted model with adequate signal(Table 4).

The particle size of different batches was found to be in the range of 112.35-277.46 nm. If other variables were maintained constant, the particle size was increased on enhancing the drug to lipid ratio(Table 1, batch F1 237.05 nm and F2 259.57 nm). This might be due to the aggregation of the particles due to the insufficient surfactant to disperse the particles. On the other hand, surfactant(1.5-4.5%)exhibited a negative effect on the particle size(Table 1, batch F1 237.05 nm and F3 112.35 nm). This might be due to a decrease in interfacial tension between the aqueous and lipid phase which prevents particle aggregation). Homogenization speed exhibited a negative impact (Table 1, batch F5 187.46 nm and F7 134.27 nm)on the particle size due to the generation of high force which breaks the particles and hence reduces the particle size. Equation 3 indicated that the effect of surfactant(coefficient value-40.87)exhibited a more prominent effect on particle size as compared to homogenization speed(coefficient value -24.97). cistanche deserticola side effects Figure 2 explored the influence of various independent parameters on particle size.

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3.4 Effect of certain parameters on entrapment efficiency(Y2)

The following polymer equation was obtained from BBD showing the influence of different independent parameters on the entrapment efficiency:

Here， variables A， B， C， AB， AC， BC， A， and B²had s a significant influence on entrapment efficiency. At a 95%confidence interval, the lack of fit was insignificant(p<0.05)while the remaining parameters were found to be significant(p<0.0001)with adequate precision(>4)(Table 3). The quadratic model（R²=0.9991）was considered as the best-fitted model with an adequate signal(Table 4).

From the above equation 4, it is observed that lipid (compritol 888ATO)exhibited a positive effect while the remaining two parameters exhibited a negative influence on entrapment efficiency. The surfactant(coefficient value -7.23)had a more prominent effect as compared to homogenization speed(coefficient value -5.76). The positive effect of lipid on entrapment efficiency(Table 1, batch F1 and F2)was due to the availability of more lipid for the accommodation of available drugs. A variable effect was observed on entrapment efficiency. At the initial stage, on increasing the surfactant, the value of entrapment efficiency was increased but on further increasing the surfactant, it was decreased due to leakage of the drug in the external environment(Table 1, batch F1 and F3). Homogenization speed exhibited a negative effect on entrapment efficiency due to more shear force which might responsible for the expulsion of the drug(Table 1, batch F5 and F7). Figure 3 explored the effect of various independent parameters on

3.5 Influence of independent parameters on PDI(Y3)

Following polynomial equation showing the influence of various parameters on the PDI:

Here,variables like A,B, C,AB,AC,BC,A2,and C°had a significant effect on the PDI. At a 95% confidence interval, the lack of fit was insignificant(p>0.05)while the remaining parameters were found to be significant(p<0.0001)with adequate precision(>4)(Table 3). The quadratic model(R=0.9991)was considered as the best-fitted model with an adequate signal(Table 4).

From equation 5, it is stated that all the three independent parameters like a drug lipid ratio, surfactant, and homogenization speed had a positive effect on the PDI. The dominant effect was due to homogenization speed(co-efficient value 0.048)followed by lipid(coefficient value 0.046)and the least was surfactant(coefficient value 0.014).Due to homogenization, the kinetic energy of the system became somewhat high which cause the collision and aggregation of lipid nanoparticles(Table 1, Batch F9 and F11). A high concentration of surfactant produced more small particles that form a bridge with big particles and hence produce non-uniformity which enhances PDI. Increasing the lipid concentration at a constant surfactant level caused the coagulation of particles resulting in a non-uniform particle size distribution. Figure 4 explored the influence of various independent variables on the PDI). Based on three characteristics i.e. particle size, entrapment efficacy, and PDI the batch F9 was considered an optimized formulation with the value of particle size, entrapment efficiency, and PDI 277.46 nm,80.48%, and 0.275 respectively.

3.6 Characterization of OLP-SLNs

3.6.1 Evaluation of particle size, PDI, and zeta potential

The value of particle size, PDI, and zeta potential of the optimized batch(F9)was found to be 277.46 nm, 0.282, (Fig.5A), and -23.18 mV respectively. The small value of PDI(<0.5)indicates the uniform or mono distribution of particles without any aggregation in the developed SLNs dispersion. A similar finding was observed by Yasir et al. cistanche dosage reddit during the production of Buspirone-loaded SLNs for nose to brain delivery. Both particle size and surface charge are important in the case of nanoparticle drug delivery. The particle size less than 500 nm is thought to be escaping from the phagocytosis mechanism induced by macrophages. Zeta potential in the form of a negative surface charge(around -20 mV)is desirable for the proper stability of nano-formulation. The observed value of zeta potential was -23.18 mV indicating good physical stability

of SLNs dispersion. An important role of negative surface nanoparticles is their attraction to the positively charged proteins from damaged tissues and help in regulating the oxidation process". The developed optimized formulation

(F9)was fulfilling both criteria of particle size and surface charges.

3.6.2 Morphological study

The optimized batch of developed OLP-SLNs(F9)indicates the roughly spherical shape as observed by the TEM study(Fig.5B).

3.6.3 Entrapment efficiency(%)

The entrapment efficiency of developed batches of OPL SLNs was found to be in the range of 50.17-86.46% with 80.48% of optimized formulation(F9)indicating a good drug entrapment ability of developed SLNs. 3.6.4 Differential scanning calorimetry(DSC)study Properties like crystallinity and thermal behavior of the developed OLP-SLNs are important properties that ensure their application in drug delivery. The DSC thermogram of OLP(drug),lipid(compritol 888 ATO),and optimized formulation(F9)is represented in Fig. 6. The thermal spectrum of OPL showed a short and broad endothermic peak at 65.23℃ but it went up to 100.96℃. The compritol 88ATO showed an endothermic peak at 70.5℃ which re-sembling with its melting point. The characteristics peak of OLP was absent in the thermogram of optimized formulation(F9). Here, only one comparatively broad peak around 67.81℃ was observed signifying the entrapment of OLP in the lipid matrix leading to the formation of OLP-SLNs4.

3.6.5 In-vitro drug release

The release of drug from optimized OLP-SLNs formulation(F9)was found to be 95.29±8.13% as shown in Fig.7. The release from the optimized formulation exhibited a bi-

phasic pattern ie. initial fast release(23.83±4.51 in first 1 h)due to the release of surface absorbed drug and later sustained(95.29±8.13% in 24 h)due to drug release from SLNs matrix. The release kinetic of OLP form optimized formulation was detected by putting the obtained drug release data into the different kinetic models as represented graphically in Fig.8. The maximum R²（0.9984）value was found to be for the first-order kinetic model. Therefore, first-order kinetic was considered the best-fitted model. The release mechanism was found to be the Fickian diffusion type with the value of release exponent(n)0.441.

3.6.6 Stability evaluation

Under the proper storage conditions, any developed dosage form is expected to be stable up to its duration of usage(expiry date). Here, the developed optimized OLP-SLNs formulation(F9)was stored at certain storage conditions as per the specification provided by ICH guidelines. The formulation stored at 4±2℃(refrigerator)and room conditions(25±2℃/60±5% RH)were not significantly(p<0.05) differ from the initial data(zero time)in respect of particle size, PDI, surface charge(zeta potential) and entrapment efficiency. A significant reduction(p<0.05)in zeta potential(-19.27 mv)and entrapment efficiency (73.29%)and a significant increment(p<0.05)in particle size(388.37 nm)was observed in the formulation stored at 40±2℃/75±5% RH.It might be due to the partial loss of surfactant covering(hence zeta potential reduced)which leads to aggregation of particles(hence particle size increased)and leakage of a drug in the external environment (hence entrapment efficiency reduced)4,4,46)

3.6.7 Antioxidant activity

The main action of OLP is to prevent peroxidation by cleaning free radicals and contributing to the assuagement of injuries caused by oxidative stress. As shown in Fig. 9, free OLP exhibited 48.38±5.28 5% anti-oxidant which is significantly(p<0.01)less than the anti-oxidant activity (67.93±7.37%)of optimized formulation(F9). This could be due to the nano-size of the lipid particles which offered a higher surface area for the chemical quenching and also protect the OLP in the external environment. No absorbance was found for the blank SLNs and hence blank SLNs did not exhibit any radical scavenging activity. Similar findings were reported previously". The value of anti-oxidation activity for ascorbic acid was supposed to be 69.42±5.38% which was not significantly different(p<0.05)from the optimized formulation(F9). 3,6.8 Anti-microbial study

The study exhibited the anti-microbial potential of developed SLNs formulation of OLP(F9)against the bacteria like Staphylococcus aureus(Gram's positive)and Pseudomonas aeruginosa(Gram's negative)as shown in Fig.10 The result exhibited that there was no zone of inhibition for blank formulation as it was free from the OLP extract.

The maximum zone of inhibition for positive control and physical mixture(OLP extract+blank SLNs)was observed within the first 6h. After this duration(first 6h), there was no change in the zone of inhibition of positive control and physical mixture. The value of the zone of inhibition for positive control against Pseudomonas aeruginosa and Staphylococcus aureus was 7.5±1.25 mm and 8±1.30 mm respectively. Similarly, the value of zone of inhibition for the physical mixture(OLP extract+blank SLNs)against Pseudomonas aeruginosa and Staphylococcus aureus was found to be 8.25±1.9 mm and 8.60±2.1 mm respectively. On the other hand, OLP-loaded SLNs exhibited the anti-microbial effect for a longer time(up to 24 h)as compared to free OLP and physical mixture. This is due to the sustained release of OLP from SLNs formulation. The antimicrobial effect of OLP-loaded SLNs against both gram-positive

and gram-negative bacteria were observed for up to 24 h. The antimicrobial effect of OLP-SLNs was significantly(p <0.001)more than that of OLP extract and physical mixture against Pseudomonas aeruginosa and Staphylococcus aureus. The value of the zone of inhibition of OPL-SLNs against Pseudomonas aeruginosa and Staphylococcus aureus was observed at 14.75±2.25 mm and 16.30±2.1 mm in 24 h respectively. Moreover, study findings indicated that all formulations containing OLP extract exhibited better anti-microbial efficiency towards Gram-positive Staphylococcus aureus as compared to Gram-negative Pseudomonas aeruginosa,4". To the best of our knowledge, the previous report showed that the major constituents for antimicrobial activity present in the olive extract are Cyclotrisiloxane hexamethyl(36.98%), Cyclo-tetrasiloxane octamethyl(15.18%), and Cyclopentasilox-ane desmethyl(14.59%)2.

4 Conclusion

In this study, olive leaves extract powder which was less stable in normal environmental conditions was successfully converted into a stable SLNs formulation. The optimized formulation exhibited a promising particle size, entrapment efficiency as well as surface charges. The optimized formulation exhibited a sustained drug release pattern up to 24 h following the first order drug release kinetic and Fickian diffusion type of drug release mechanism. The stability study was conducted and the optimized formulation(F9)was stable under the stated storage conditions. OLP formulation exhibited a promising anti-oxidant property as justified by the DPPH assay method. The antimicrobial activity against Gram's positive(Staphylococcus aureus)and Gram's negative(Pseudomonas aeruginosa)bacteria. Finally, it was concluded that SLNs could be the promising carriers for the delivery of olive leaf extract powder.

This article is extracted from J. Oleo Sci. 70, (10) 1403-1416 (2021)